Selenium is an essential element in the diet of mammals and too little or too much in the diet within a narrow range can have devastating effects on health. Selenium has a role in preventing heart disease and has been implicated in preventing cancer and in delaying the aging process. This element exerts its effects, in part, through its presence in selenoproteins as the amino acid, selenocysteine. We, therefore, are focusing on the means by which selenocysteine is biosynthesized on tRNA and is incorporated into protein. In the past year, we have used targeting vectors to remove one copy of the selenocysteine tRNA gene from the genome of mouse embryonic stem cells. The selenocysteine tRNA population is reduced approximately 50% in these cells, but the biosynthesis of selenoproteins apparently is unaffected suggesting that the levels of the selenocysteine tRNAs are not limiting in protein synthesis. Knockout mice are being developed from the mouse embryonic stem cells lacking the tRNA gene to examine the effects of the loss of this gene on mice. We are also making transgenic mice with extra copies of the selenocysteine tRNA gene in their genome to study the effects of an enriched selenocysteine tRNA population on protein synthesis. We have completed a study on an analysis of the selenocysteine tRNA population in selenium deficient rats which have been resupplemented with selenium. The latter studies show that an enrichment of the selenocysteine tRNAs in the presence of selenium and an alteration in the distribution of the corresponding isoacceptors occurs and that the rate of enrichment and change is tissue dependent. A new selenoprotein, designated as 15 kDa protein, was discovered and partially purified by one member of our laboratory prior to his coming here. Since his arrival, the 15 kDa protein has been sequenced, it has been localized on human chromosome 1 and evidence suggests that it may be involved in the progression of cancer. In addition, large quantities of the selenocysteine tRNA isoacceptors and of serine tRNA have been prepared to analyze crystals of these molecules complexed with seryl-tRNA synthetase. Seryl-tRNA synthetase aminoacylates both selenocysteine and serine tRNAs. Drosophila selenocysteine tRNAs were isolated in large quantities and their primary sequences determined. These tRNAs are co-linear with the corresponding gene. Analysis of the selenocysteine tRNA population in bovine liver and testis and Chinese hamster liver and ovaries have shown that these isoacceptors migrate differently on polyacrylamide gels in liver and testis and in Chinese hamster liver and ovaries suggesting that the different species may arise by editing. We are further investigating this possibility. Transcription of this gene has revealed a new factor involved in expression of Pol III genes containing a TATA box.